The objectives of the proposed research are to understand the roles of myosin I in membrane retraction and retrograde actin flow. Myosin Is represent one of the largest classes of unconventional myosins in humans, yet little is known about their activity, distribution, or regulation in vertebrate cells. Specific aims include the following: 1) To characterize the distribution of myo1c during lamellipodial extension and retraction in MTLn3 cells, a metastatic mammalian cell line. This will be accomplished by immunofluorescence microscopy and transient expression of eGFP-myosin fusion proteins. 2) To determine the effect of myosin-I "knockdown" on cell spreading, membrane retraction, and retrograde actin flow using small interfering RNA (siRNA). Retrograde flow in control and myosin-I knockdown cells will be assessed by motility of surface-attached latex beads. 3) To determine the relationship between the mechanical activity of myo1c and membrane dynamics. We will express myo1c mutants that have altered motor properties, and we will examine the effect of these mutants on membrane dynamics.